Liquid Biopsy for Proliferative Diabetic Retinopathy: Single-Cell Transcriptomics of Human Vitreous Reveals Inflammatory T-Cell Signature

Abstract

Purpose: Current therapies for proliferative diabetic retinopathy (PDR) do not specifically target VEGF-independent, cell-type-specific processes that lead to vision loss, such as inflammatory pathways. This study aimed to identify targetable cell types and corresponding signaling pathways by elucidating the single-cell landscape of the vitreous of patients with PDR.

Design: Case series.

Subjects: Vitreous and peripheral blood obtained from 5 adult patients (6 eyes) undergoing pars plana vitrectomy for vision-threatening PDR.

Methods: Single-cell RNA sequencing (scRNA-seq) was performed on vitreous cells obtained from diluted cassette washings during vitrectomy from 6 eyes and peripheral blood mononuclear cells (PBMCs, n = 5). Droplet-based scRNA-seq was performed using the Chromium 10x platform to obtain single-cell transcriptomes. Differences in tissue compartments were analyzed with gene ontology enrichment of differentially expressed genes and an unbiased ligand-receptor interaction analysis.

Main outcome measures: Single-cell transcriptomic profiles of vitreous and peripheral blood.

Results: Transcriptomes from 13 675 surgically harvested vitreous cells and 22 636 PBMCs were included. Clustering revealed 4 cell states consistently across all eyes with representative transcripts for T cells (CD2, CD3D, CD3E, and GZMA), B cells (CD79A, IGHM, MS4A1 (CD20), and HLA-DRA), myeloid cells (LYZ, CST3, AIF1, and IFI30), and neutrophils (BASP1, CXCR2, S100A8, and S100A9). Most vitreous cells were T cells (91.6%), unlike the peripheral blood (46.2%), whereas neutrophils in the vitreous were essentially absent. The full repertoire of adaptive T cells including CD4+, CD8+ and T regulatory cells (Treg) and innate immune system effectors (i.e., natural killer T cells) was present in the vitreous. Pathway analysis also demonstrated activation of CD4+ and CD8+ memory T cells and ligand-receptor interactions unique to the vitreous.

Conclusions: In the first single-cell transcriptomic characterization of human vitreous in a disease state, we show PDR vitreous is primarily composed of T cells, a critical component of adaptive immunity, with activity and proportions distinct from T cells within the peripheral blood, and neutrophils are essentially absent. These results demonstrate the feasibility of liquid vitreous biopsies via collection of otherwise discarded, diluted cassette washings during vitrectomy to gain mechanistic and therapeutic insights into human vitreoretinal disease.

Financial disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Keywords: Diabetic retina; PDR; PPV; Pars plana vitrectomy; Proliferative diabetic retinopathy; Single cell; Vitreous.

Figure 1

Single-cell atlas of the human vitreous and peripheral blood mononuclear cells (PBMCs) in individuals with proliferative diabetic retinopathy (PDR) A, Schematic diagram of study design with sample collection from vitreous and PBMCs from each human donor undergoing vitrectomy for proliferative diabetic retinopathy (PDR). Uniform Manifold Approximation and Projection (UMAP) of integrated scRNA-seq analysis of the vitreous (13 675 cells) and PBMCs (22 636 cells) from all 6 eyes resulting in 36 311 cells in total. Major immune cell populations are shown in (B) with comparison of these populations between the vitreous and peripheral samples in (D). The retrospective distribution of major immune cell populations by proportion (C) and cell numbers (E) per donor eye. F, Dot plot of differentially expressed genes in each identified cluster and associated immune cell population. This figure was created with BioRender.com.

Figure 3

Distinct effector profiles of T-cell populations within vitreous and PBMC populations from individuals with PDR. A, UMAP plot of the cluster identifications for effector T-cell populations and their cytotoxic profiles and (B) respective UMAP plot of cell location from the vitreous and peripheral blood. (C) Dot plot of differentially expressed genes in each identified cluster and effector T-cell population. Comparison of (D) cell counts and (E) proportion of T cells per cluster in vitreous and PBMC tissues. Enriched gene ontology biological processes found in (F) CD4 memory T cells and (G) CD8 memory T cells when comparing vitreous to PBMCs. NK = natural killer; MAIT = mucosal-associated invariant T; Treg = regulatory T cell; FC = fold change; FDR = false discovery rate; PBMC = peripheral blood mononuclear cell; PDR = proliferative diabetic retinopathy; UMAP = Uniform Manifold Approximation and Projection.

Figure 5

Myeloid-lineage cell and B-cell populations within vitreous and PBMCs from individuals with PDR. A, UMAP plot of the cluster identifications for macrophage and dendritic cell populations and (B) respective UMAP plot of cell location from the vitreous and peripheral blood. (C) Dot plot of differentially expressed genes in each identified cluster and monocyte population. D, E, Comparison of myeloid cell subtypes per cluster based on cell location by cell numbers and proportion. F, UMAP plot of the cluster identifications for B-cell populations and (G) respective UMAP plot of cell location from the vitreous and peripheral blood. (H) Dot plot of differentially expressed genes in each identified B-cell cluster. I, Comparison of cell numbers and (J) cell proportion of B-cell type per cluster based on cell location. IFN = interferon; Mono = monocyte; Mac = macrophage; DC = dendritic cell; FC = fold change; FDR = false discovery rate; PDR = proliferative diabetic retinopathy; UMAP = Uniform Manifold Approximation and Projection.

Figure 6

Cell–cell interactions within the vitreous and PBMC compartments. A, Heatmap representing cell–cell interactions identified within the vitreous compartment using Dimer Signal Receptor Analysis (DiSiR) which predicts ligand–receptor interactions at the cell-type level using the scRNA-seq data. Similarly, cell–cell interactions within the PBMC compartment were identified (B) with the data presented limited to interactions also possible in the vitreous compartment. C, Cell–cell interactions more prominent in the vitreous compartment are highlighted in this heatmap. PBMC = peripheral blood mononuclear cell.